CRISPR-based genetic tools for the study of host-microbe interactions

Abstract

CRISPR-based genetic tools have revolutionized our ability to interrogate and manipulate genes. These tools can be applied to both host and microbial cells, and their use can enhance our understanding of the dynamic nature of host-microbe interactions by uncovering their genetic underpinnings. As reviewed here, CRISPR-based tools are being used to explore the microbiome in an efficient, accurate, and high-throughput manner. By employing CRISPR screens, targeted genome editing, and recording systems to the study of host cells and microorganisms, we can gain critical insights into host defense mechanisms, potential vulnerabilities, and microbial pathogenesis, as well as essential or condition-specific genes involved in host-microbe interactions. Additionally, CRISPR-based genetic tools are being used in animal models to study host-microbe interactions in vivo. Recent advancements in CRISPR-derived technology can be combined with emerging techniques, such as single-cell RNA sequencing, to examine the complex interactions between hosts and microbes, shedding light on the role of the microbiome in health and disease. This review aims to provide a comprehensive overview of how these cutting-edge genetic tools are being used to investigate host-microbial systems, as well as their current limitations. Current research is likely to yield even more advanced genetic toolkits than those presently available, and these can serve researchers in identifying and exploring new therapeutic targets for diseases related to host-microbe interactions.

Keywords: CRISPR; genetic tools; host-microbe interactions.

Fig 1

Overview of traditional and CRISPR-based methods for studying host-microbe interactions. Traditional methods (left) include germ-free (GF) mouse models, fecal microbiota transplantation (FMT), and colonoscopy. CRISPR-based methods (right) feature the design of whole-cell biosensors, transcriptional recorders, and CRISPR screens for gene characterization.

Fig 2

Application of CRISPR methods for studying host-microbe interactions. (A) CRISPRi-based genetic circuits have been designed for precise, stimulus-responsive gene inhibition in B. thetaiotaomicron in the murine gut (49). (B) Record-seq, a CRISPR-based molecular recording method, has been used to design transcriptional recorders that detect diet- and inflammation-specific transcription signals in vivo (72). (C) CRISPRi genome-wide screening in antibiotic-resistant M. tuberculosis revealed novel vulnerabilities (54). (D) Genes improving the survival of THP-1 human phagocytic cells infected by Mycobacterium bovis were identified by whole-genome CRISPR-KO/CRISPRi screening (76). DSS, dextran sodium sulfate.

Fig 3

Strategies for the in situ delivery of CRISPR tools to complex microbial communities. (A) Phages provide highly specific gene delivery, infecting only susceptible bacterial hosts. (B) Engineered phages infect Clostridioides difficile, depleting these bacteria in the murine gut by inducing genome degradation through a heterologous CRISPR array, with contributions from endogenous Cas3 and Cascade complexes (112). (C) Engineered phages deliver base editors into commensal bacteria in the murine gut microbiota, enabling precise in situ gene editing (113). (D) Bacterial conjugation facilitates low-specificity DNA delivery, wherein donor bacteria transfer a conjugative plasmid containing the desired genetic payload to recipients. (E) Probiotic donor bacteria deliver a CRISPR-Cas9 system specifically targeting antibiotic resistance genes, rendering antibiotic-resistant bacterial strains in the murine gut microbiota sensitive to antibiotics (114). (F) Probiotic conjugation donors transfer a CRISPR-Cas Tn7 complex, facilitating the insertion of transposons into specific bacteria in an infant’s fecal microbiota (115).